Methods for characterizing RNAs generally require the attachment by ligation of adapters of known sequence to the 3′ end and to the 5′ end of a target RNA. The ligation reactions are preferably performed in sequence and utilize an excess of the adapters. An undesirable consequence of the ligation reaction is the formation of dimers consisting of the 3′ adapter and the 5′ adapter which in subsequent reactions involving cloning or amplification give rise to significant background.
Degradation of the excess 3′ adapters has been achieved enzymatically. For example, Pease, Nature Methods, 8: iii-iv (2011), and Vaidyanathan, et al., United States Patent Application US 2011/0104785 A1, describe the use of nucleases after individual ligation steps. However, the enzyme must be inactivated prior to addition of a 5′ adapter for ligation to avoid its degradation during subsequent ligation steps.
Another approach relies on a spanning oligonucleotide which is complementary to the junction of the adapter dimer. The spanning oligonucleotide is used to prevent the adapter dimer from being copied into ssDNA by reverse transcriptase (Kawano et al., Biotechniques 49(4): 751-755 (2010)). Unfortunately, design constraints are imposed on the sequence of the spanning oligonucleotide. The spanning oligonucleotide must block cDNA synthesis of the adapter dimer but not block cDNA synthesis of the ligated target RNA. Moreover, formation of the dimer is not blocked. Instead, the method prevents the dimer from being copied into a second strand. An adverse consequence of using a spanning oligonucleotide is that a majority of the 5′ adapter and the ligase in the second ligation are used for formation of the oligonucleotide dimer, not for ligation to the target RNA which reduces the yield of ligated target RNA.
A standard approach to reducing high background is described by Lau et al., Science 294:858-862 (2001), who used gel purification of the first ligation product to remove excess 3′ adapter that was not ligated to the RNA. However, this approach preferentially uses reduced amounts of the adapters to reduce dimer formation and this in turn reduces the yield of the desired product for cloning or sequencing.